Use of the spreadsheet method incorporating students' t-test analysis to examine hyaluronan binding function of wild type and mutant CD44 receptors expressed in AKR1 cells AKR1 cells transfected with WT CD44 or CD44-T were stained for three-colour flow cytometry as described in the Methods. For the negative control, WT CD44 expressing cells were stained with FITC-BSA and rabbit anti-mouse PE-F(ab)2. The flow cytometry data is displayed as follows: A, histogram plots showing CD44 expression or FITC-hyaluronan binding. B, two-parameter dot plots correlating FITC-hyaluronan binding against CD44 expression. C, the data was analysed using the spreadsheet method (see text for further details). Where there were 3 cells or fewer for a given PE fluorescence channel, the point is plotted at 0 on the y-axis. Values are arbitrary fluorescence units. Analysis used the spreadsheet method including application of a students' t-test. Where there was a significant statistical difference in FITC-hyaluronan binding between WT CD44 and CD44-T at the 99.9% level, a point is plotted at 980 on the y-axis.